The present invention relates to a method for producing livestock individuals from cells of an established cell line, precisely to a method for producing cloned livestock from cells of a uniform cultured cell strain (i.e. an established cell line) that serve as donor cells for nuclear transplantation. More precisely, the invention relates to a method for producing cloned cattle from cells of an established bovine intramuscular preadipocyte (hereinafter referred to BIP), a type of the uniform cultured cell strain for donor cells for nuclear transplantation.
Heretofore, cloned livestock have been produced through nuclear transplantation of non-uniform somatic cells serving as donor cells. Concretely, cells, of which the origin is not clear, are sampled at random from ears or proligerous membranes (cumulus oophorus), and these are used for nuclear transplantation.
In thus method, however, it is extremely difficult to all the time efficiently produce cloned livestock even though the same experimental condition is repeated and continued.
In addition, even when the nucleus of a somatic cell is transplanted into an egg cell, it is impossible to accurately analyze the mechanism of its cleavage like that of fertilized eggs.
Heretofore, it has been recognized that fresh living cells are necessary for producing cloned livestock. For this, therefore, the general method is nuclear transplantation of somatic cells of specific livestock, as so mentioned hereinabove.
However, for all the time efficiently producing cloned livestock, it is necessary to culture cells of the same types applicable to cloned livestock production and to establish the cell line.
To solve the problems as above, we, the present inventors have assiduously studied a technique of culturing intramuscular fat cells of livestock, especially those of cattle, and a method of establishing the cell line, and, as a result, have succeeded in establishing a bovine intramuscular preadipocyte (BIP) usable for donor cells for nuclear transplantation. We have analyzed the established cell line as to whether or not it is suitable for donor cells for nuclear transplantation, and, as a result, have found that the BIP is useful for producing cloned cattle. On the basis of these findings, we have completed the present invention.
Hereinafter, the present invention will be described in detail.
The present invention relates to a method for producing cloned livestock comprises using a cell of uniform cultured cell strain as a donor cell for nuclear transplantation. That is to say, in a method for producing cloned livestock, the improvement comprises using a cell of uniform cultured cell strain as a donor cell for nuclear transplantation.
More specifically, the present invention is shown as follows.
A method for producing cloned livestock, comprising;
a. preparing of recipient egg by taking cumulus oophorus-ovum complex out of ovaries; then maturing in vitro by cultivating it in a culture medium; separating and removing cumulus oophorus cells to obtain ovum; cutting a zona pellucida of the ovum; expelling a nucleus-containing cytoplasm to obtain enucleated cell
b. transplanting a uniform cultured cell strain as a donor cell into a perivitelline space of the enucleated matured ovum
c. transferring said ovum with the donor cell into a cell fusion medium, and then giving an electric shock whereby the donor cell is fused with the ovum to obtain nucleus-transplanted ovum
d. culturing said nucleus-transplanted ovum to make it have a germinated blastocyst, and then selecting a good embryo
e. transplanting the good embryo into a surrogate livestock, and
f. checking whether said embryo get pregnant, and further monitoring the condition of fetus.
Following are embodiments of the present invention.
The method as claimed above, wherein the livestock is a livestock selected from the group consisting of cattle, sheep, goats and pigs.
The method as claimed above, wherein the cell of uniform cultured cell strain is a BIP.
The method as claimed above, wherein a cryopreserved cell is utilized as the bovine intramuscular preadipocyte.
In the method of the invention, BIP can be preferably used as the uniform cultured cell strain. The livestock to which the method of the invention is applicable include, for example, cattle, sheep, goats and pigs. Of those, especially preferred are cattle.
BIP can be established as follows: Fat cells and preadipocytes (fat precursor cells) are separated from bovine musculi longissimus thoracis with marbling (fat tissue) therein, and these are cultured for primary culture. Concretely, the marbling part is collected from the musculi longissimus thoracis of a slaughtered cow, and the fat tissue is separated from it. The fat tissue is analyzed for the presence or absence of cell growth therein, and the growth is only observed in the preadipocytes. The fat tissue is treated with enzyme to collect the preadipocyte.
Next, the preadipocytes are sub-cultured and cloned according to an ordinary method, and a single BIP cell line having the ability to grow is thereby established.
The cells of the BIP cell line are analyzed, and it is found that they are particular cells of pericytes existing around blood vessels. Up to the present, no one has succeeded in establishing the cell line of the pericytes.
One embodiment of the invention is described below, which is for producing cloned livestock from BIP serving as donor cell for nuclear transplantation.
The method of producing cloned livestock may be basically the same as the conventional method of producing them from somatic cell serving as donor cell. Concretely, a cumulus oophorus-ovum complex (COCs) is taken out of the ovary of a slaughtered cow, and this is matured in vitro. The cumulus oophorus cells are separated and removed from the COCs, and the nucleus is removed from the resulting ovum. BIP cell is transplanted into enucleated ovum, and these are fused with an electric pulse imparted thereto to make a nucleus-transplanted ovum.
Next, the nucleus-transplanted ovum is cultured to generate a blastocyst. From those, good embryos are selected, and each is transplanted in a surrogate cow. The surrogate cows with the embryos transplanted are checked as to they get pregnant or not. While the condition of the fetus therein is monitored, each surrogate cow is raised, and the birth of a cloned calf from it is waited for.
Since the BIP cells are of a cloned uniform cell line, their properties are stable, and all generations of the cells can be used for nuclear transplantation. Concretely, the present inventors"" experiments have shown that the cells can be sub-cultured up to at least 60 generations with no chromosomal aberrations in at least these 60 generations of the sub-cultured cells, and that there is no change of the rate of blastocyst generation in the nuclear transplantation of the sub-cultured cells.
The BIP cells can be cryopreserved for storage in any known manner. The cryopreserved cells can be thawed anytime before use. In addition, the BIP cells can easily undergo any desired gene introduction or deletion. Therefore, for example, it is possible to produce cattle that are not infected with bovine spongiform encephalopathy (BSE).
According to the invention, cloned livestock can be stably and efficiently produced. In addition, since the donor cells used in the invention are of a uniform cultured cell strain, they can be cryopreserved, and can be thawed before use. Therefore, the same donor cells can be cultured anytime when necessary. Moreover, specific genes can be easily introduced into or deleted from the donor cells through a technique of genetic engineering, and, in addition, it is possible to confirm the gene introduction or deletion after the genetic modification.